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Indian J Microbiol (Jan–Mar 2014) 54(1):114–117

DOI 10.1007/s12088-013-0438-4


Azithromycin Reduces the Production of a-hemolysin and Biofilm

Formation in Staphylococcus aureus
Zhihong Gui • Huafu Wang • Ting Ding •

Wei Zhu • Xiyi Zhuang • Weihua Chu

Received: 29 August 2013 / Accepted: 9 November 2013 / Published online: 21 November 2013
Ó Association of Microbiologists of India 2013

Abstract Staphylococcus aureus causes a broad range of these environments and causes potentially fatal infections
life-threatening diseases in humans. This bacterium pro- in immunocompromised hospital patients. S. aureus cause
duces a large number of extracellular virulence factors that disease through the production of virulence factors. These
are closely associated with specific diseases which are factors include adhesins, exotoxins, enterotoxins, hemoly-
controlled by quorum sensing. In this study, we show that sins, and leukocidin, as well as proteases that enable the
azithromycin was active against methicillin-resistant bacteria to spread within the host [2, 3]. Quorum sensing
Staphylococcus aureus (MRSA) strains with MICs ranged (QS) regulates expression of genes encoding these viru-
from 32 to 64 lg/mL. Azithromycin at subinhibitory con- lence factors and biofilm formation. Strains defective in
centration, markedly reduced the production of a-hemol- their ability to form a biofilm or produce toxins show
ysin at (1/16MIC, 1/8MIC) and biofilm formation at diminished virulence [4], suggesting that a novel approach
(1/16MIC, 1/8MIC), respectively. The results indicated for therapy development would be to interfere with the
that sub-inhibitory concentrations of azithromycin production of virulence factors [5–7]. Azithromycin
decreased the production of a-hemolysin and biofilm for- (AZM) is one of the macrolides antibiotics, and it is a
mation in MRSA in a dose-dependent manner. Therefore, broad-spectrum macrolide antibiotic effective against
azithromycin may be useful in the treatment of a-hemol- Gram-positive, Gram-negative, and atypical bacteria and
ysin producing and biofilm formation MRSA infections. has anti-inflammatory characteristics [8]. The purpose of
this study is to assess the effect of azithromycin, used as a
Keywords Staphylococcus aureus  Azithromycin  quorum-sensing inhibitor, for decrease the production of
Biofilm  a-hemolysin QS associated virulence factors and biofilm formation in
Staphylococcus aureus.
Two different clinical bacterial strains of methicillin-
Staphylococcus aureus is found among the normal human resistant Staphylococcus aureus (MRSA) 10 and 21 were
skin flora and it is an important pathogen of humans, collected from different patients hospitalized at Lishui
causing diseases ranging from superficial skin and wound People’s Hospital, Zhejiang province. S. aureus strains was
infections to severe illnesses such as septicaemia, endo- identified biochemically from routinely obtained speci-
carditis and toxic shock syndrome [1]. S. aureus is par- mens by means of the Vitek ATB Expression System,
ticularly a problem in hospitals because it spreads easily in version 2.7.8 (BioMe’rieux Deutschland GmbH, Nürtin-
gen, Germany), which uses 32 biochemical reactions.
Bacterial isolates were stored as suspensions in LB med-
Z. Gui  H. Wang  T. Ding
Lishui People’s Hospital, Lishui 323000, China ium containing 12.5 % (vol/vol) glycerol at -70 °C until
tests were performed. S. aureus MRSA 10, 21 single col-
W. Zhu  X. Zhuang  W. Chu (&) ony was inoculated into LB media and cultured overnight
Department of Microbiology, School of Life Science and
at 37. Overnight culture was subcultured (1:100) into test
Technology, China Pharmaceutical University,
Nanjing 210009, China tubes with fresh LB medium in triplicate. Optical density
e-mail: chuweihua@cpu.edu.cn readings at 600 nm were obtained with spectrophotometer

Indian J Microbiol (Jan–Mar 2014) 54(1):114–117 115

(UV-721, Shanghai optical instrument factory, Chian) at a

30 min intervals. Experiments were repeated three times to
obtain the mean and standard error of means. Standardized
planktonic antibiotic MICs were determined by the broth
microdilution method outlined by the Clinical and Labo-
ratory Standards Institute (formerly the National Commit-
tee for Clinical Laboratory Standards) [9]. Briefly, the test
antibiotic was serially diluted twofold in tubes containing
5 mL of LB. The S. aureus inoculum for the series of tubes
was 100 lL of a 5.0 9 106 CFU/mL dilution in LB. The
MIC was the lowest concentration of antibiotic that pre-
vented turbidity after 20 h of incubation at 37 °C. For b
detection of hemolysin production, S. aureus strains were
grown in LB in the absence or presence of sub-inhibitory
concentrations of azithromycin until reaching the post-
exponential growth phase. Bacterial supernatants were
collected by centrifugation (5,0009g, 4 °C, 5 min), the
supernatant was collected, and the residual cells were
removed using a 0.2 lm filter. Prior to the addition of
25 lL of defibrinated rabbit blood, a 0.1 mL volume of
culture supernatant was brought up to a volume of 1 mL
through the addition of PBS buffer. After incubation for Fig. 1 Growth curves of S. aureus strains MRSA 10 (a) and
15 min at 37 °C, the unlysed blood cells were pelleted by MRSA21 (b) treated with or without azithromycin. (black rhombus),
centrifugation (5,0009g, room temperature, 2 min). The untreated S. aureus; (black square), S. aureus plus azithromycin at 1/2
hemolytic activity of the supernatant was detected by MIC; (black triangle), S. aureus plus azithromycin at 1/4 MIC; (*), S.
aureus plus azithromycin at 1/8 lg/mL; and (black circle), S. aureus
measuring the optical density at 543 nm. The supernatant plus azithromycin at 1/16 lg/mL
of culture without azithromycin served as the 100 %
hemolysis, and the percent hemolysis was calculated by solubilization of the crystal violet with ethanol-acetone
comparison with the control culture [10]. For screening (80:20 vol/vol) the absorbance at 590 nm was determined.
biofilm producing strains, Congo red Agar method was The MICs of azithromycin against S. aureus strains
used. This method is based on the characteristic cultural MRSA 10 and MRSA 21 were 128 and 64 lg/mL,
morphology of biofilm-forming bacteria on Congo red respectively. Growth curves for S. aureus isolates MRSA
medium. The medium was composed of brain heart infu- 10 and MRSA 21 grown in the presence of increasing
sion broth (BHI) (Oxoid Ltd, Hampshire, England) 37 g/L, concentrations of azithromycin at sub-MIC concentrations
sucrose 50 g/L, agar 10 g/L and Congo red (BDH Chem- are shown in Fig. 1. For both isolates, growth in the pre-
ical Ltd, Poole, England) 0.8 g/L. Congo red stain was sence of azithromycin at sub-MIC were not markedly dif-
made ready as a strong aqueous solution and sterilized ferent from controls. S. aureus strains were exposed to
(121 °C for 15 min) separate from the rest of the medium graded sub-inhibitory concentrations of azithromycin to the
components and supplemented to the agar when the tem- post-exponential phase. The haemolytic activities of the
perature reached 55 °C. Agar plates were prepared and culture supernatants are detected. The results shown that
inoculated and kept in the incubator for 24 h at 37 °C. The the reducing of a-hemolysin levels in S. aureus was dose-
production of black colonies with a dry crystalline con- dependent. When grown in the presence of 1/16MIC of
sistency by the organisms was taken to indicate biofilm azithromycin, the haemolysis values of 10 and 21 culture
production as non-biofilm-producing strains develop red supernatants were 63.5, 75.3 %, respectively, compared
colonies [11]. with a azithromycin-free culture Fig. 2 Notably, supple-
Biofilm formation was determined as previously mentation with 1/2MIC of azithromycin led to 15.6 and
described by Christensen et al. [12]. LB (10 mL) was 22.5 % hemolysin production. On Congo red agar plate,
inoculated with loopful of microorganism from overnight slime producing strains formed black (complete) colonies
culture plates and incubated for 24 h at 37 °C. The tubes to slightly black, whereas non producing strains develop
were decanted and washed with PBS (pH 7.3), and fixed strong red colonies. Strains MRSA 10 and 21 formed
with 2.5 % glutaraldehyde, washed once with water, and strong black pigmentation, indicated that they can form
stained with a 0.4 % crystal violet solution. After biofilm. To determine if azithromycin could be

116 Indian J Microbiol (Jan–Mar 2014) 54(1):114–117

have shown that azithromycin at sub-MIC concentrations

affects QS-regulated virulence genes and biofilm formation
in vitro [15–17] and in in vivo models of disease [18]. In
the present study, we demonstrate that sub-inhibitory
concentrations of azithromycin could dose-dependently
reduce the haemolytic activity and biofilm formation in S.
aureus. More recent studies by Parra-Ruiz et al. [19] have
shown that low concentrations of Clarithromycin could
inhibit the biofilm formation of S. aureus, but Kumar and
Ting [20] found that the biofilm formation by S. aureus was
enhanced under sub-inhibitory norfloxacin stress. Targeting
bacterial quorum sensing is an alternative strategy that is
now gaining great interest in antimicrobial therapy and
offers promising opportunities to impede pathogenesis and
Fig. 2 Relative culture supernatant hemolytic ability assay. The
its consequences without placing immediate life-or-death
culture supernatants without azithromycin served as the 100 %
haemolysis control pressure on the target bacterium [21]. However, once QSI
concentration gets below the threshold level, bacteria may
be free to express its virulence and evidence is accumu-
lating that bacteria may develop resistance to QSIs [22].
Our results showing that azithromycin significantly
decreases a-hemolysin secretion and biofilm formation in
S. aureus suggest that azithromycin may be may be useful
in the treatment of infections with MRSA S. aureus.


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